ABSTRACT Developmental signaling cascades ultimately converge in the nucleus to induce broad, yet precise, epigenomic changes that facilitate the activation of lineage-specific factors necessary for differentiation. One of the epigenomic changes frequently observed during differentiation, including myogenesis, is the limited proteolysis of the histone H3 N-terminal tail (H3NT). Although H3NT proteolysis is a common feature of diverse developmental pathways, the functional significance of this programmed epigenetic event remains largely undetermined. We recently discovered that H3NT proteolysis occurs during myoblast differentiation and that matrix metalloproteinase 2 (MMP-2) is the nuclear H3NT protease. Inhibition of MMP-2 activity impaired H3NT proteolysis concurrent with defective myogenic gene activation and myoblast differentiation. Our findings support a conceptually innovative model of a novel epigenetic pathway where programmed H3NT proteolysis at myogenic loci directly facilitates their activation necessary for proficient myogenesis. To test this model we recently pioneered the first method to identify H3-cleaved (H3cl) loci, called ChIPac-Seq. In Aim 1, ChIPac-Seq will be leveraged with established cell models to discover the loci selectively targeted for H3NT proteolysis during myoblast differentiation, which will likely include canonical, and potentially novel, myogenic loci. The necessity and sufficiency of H3NT proteolysis to facilitate myogenic loci activation and myoblast differentiation will be determined in Aims 2 and 3, respectively. In Aim 4, our previously reported histone modification and transcription factor datasets will be leveraged to examine the direct effects of H3NT proteolysis in generating specific epigenetic changes that promote H3cl loci activation during myoblast differentiation. The anticipated outcomes of this study will yield foundational insights into the mechanistic functions of H3NT proteolysis as a novel epigenetic regulator of skeletal muscle development.